The long term goal of the proposed research in Project 2 is to develop and optimize novel strategies for prophylactic immunization against the human immunodeficiency virus (HIV) using adeno-associated virus (AAV) and poxviruses as vaccine vectors. We will exploit the simian immunodeficiency virus (SIV) macaque model of AIDS as development tool. For conceptual purposes, Project 0005 can be considered in 2 parts: (A) development and testing of AAV vectors; and, (B) testing of poxvirus vectors (developed at Therion Biologics). (A) AAV is a replication defective parvovirus with unique features that make it attractive as a vector for the stable delivery of foreign DNA (in this case, HIV genes) to cells, and overall, as a vaccine vehicle. First, AAV does not cause any disease in the infected host (safety). Second, AAV integrates in a site-specific fashion on chromosome 19 (long- term expression). Third, AAV infects most (if not all) mammalian cells (broad tropism). Fourth, AAV is extremely stable and heat resistant (cold chain not required). Finally, cloned AAV DNA proviruses are infectious after transfection and can be easily manipulated in vitro (expression systems can be optimized). The specific objectives of this research are to: (i) derive and characterize AAV vectors that direct expression of SIV/HIV genes; (ii) generate and characterize rAAV particles in vitro; (iii) test immunogenicity and efficacy of rAAV/SIV in mice and macaques; (iv) develop new strategies for the efficient packaging of rAAV; and, (v) generate rAAV/HIV-1 for testing in non-human primate models of HIV-1 infection. (B) The goal of the second part of Project 0005 is to build on work previously performed under the auspices of this NCVDG. Specifically, we will continue to investigate strategies using live recombinant vaccinia virus vectors (rVV) and pseudovirions (non-infectious SIV particles generated from rVV in cell culture). In addition, we will test new rVV which express auxiliary SIV/HIV genes (nef, vif, vpx) and novel attenuated poxvirus vectors. Lastly, we will explore sequential immunizations with poxvirus vectors and rAAV. By combining a number of unique approaches to HIV immunization, we will gain invaluable knowledge concerning the optimal approach and composition for a safe and effective HIV vaccine.